The VEGFR/PDGFR tyrosine kinase inhibitor, ABT-869, blocks necroptosis by targeting RIPK1 kinase

Necroptosis is a mode of programmed, lytic cell death that is executed by the mixed lineage kinase domain-like (MLKL) pseudokinase following activation by the upstream kinases, receptor-interacting serine/threonine protein kinase (RIPK)-1 and RIPK3. Dysregulated necroptosis has been implicated in the pathophysiology of many human diseases, including inflammatory and degenerative conditions, infectious diseases and cancers, provoking interest in pharmacological targeting of the pathway. To identify small molecules impacting on the necroptotic machinery, we performed a phenotypic screen using a mouse cell line expressing an MLKL mutant that kills cells in the absence of upstream death or pathogen detector receptor activation. This screen identified the vascular endothelial growth factor receptor (VEGFR) and platelet-derived growth factor receptor (PDGFR) tyrosine kinase inhibitor, ABT-869 (Linifanib), as a small molecule inhibitor of necroptosis. We applied a suite of cellular, biochemical and biophysical analyses to pinpoint the apical necroptotic kinase, RIPK1, as the target of ABT-869 inhibition. Our study adds to the repertoire of established protein kinase inhibitors that additionally target RIPK1 and raises the prospect that serendipitous targeting of necroptosis signalling may contribute to their clinical efficacy in some settings.


Introduction
Necroptosis is a caspase-independent mode of regulated cell death that is morphologically characterised by cell swelling, plasma membrane rupture, and the spillage of the cellular contents into the extracellular milieu initiating an inflammatory response [1][2][3]. Ancestrally, necroptosis is thought to have evolved as an altruistic host defence pathway, which is reflected by convergent evolution of analogous pathways in plants and fungi [4][5][6], and the discovery of pathogen-encoded protein inhibitors of the pathway [7][8][9][10]. Contemporary interest in the pathway has been driven by the implication of errant necroptosis in the pathophysiology of inflammatory diseases [11], such as those of the skin [12], gut [13], kidney [14,15] and lung [16], which has prompted much interest in targeting the pathway therapeutically.
While necroptosis signalling is tightly regulated in human cells [25,34,35], our previous studies revealed that mutations in the pseudokinase domain can convert mouse MLKL into a constitutively lethal form [20]. We observed that expression of mouse MLKL harbouring mutations to emulate RIPK3-mediated phosphorylation of the MLKL pseudokinase domain activation loop, or of adjacent residues in the ATP-binding site, leads to constitutive cell death upon expression alone, in the absence of upstream necroptotic stimuli [20]. Using one of these constitutively activated mouse MLKL mutants -Q343Aexpressed in mouse dermal fibroblasts (MDFs), we established a cell-based phenotypic screen to identify small molecules that modulate necroptosis [36,37]. This approach previously identified two inhibitors of necroptosis, 17-AAG [36] and AMG-47a [37], which target the chaperone, HSP90, and the kinases, RIPK1 and RIPK3, respectively. Here, we identify ABT-869a small molecule previously described as an inhibitor of tyrosine kinases of the VEGF and PDGF receptor families [38] as a novel inhibitor of necroptosis. Using biochemical, biophysical and cellular studies, we characterise the apical necroptotic kinase, RIPK1, as a previously unrecognised target of ABT-869. In concert with other reports, our findings raise the possibility that off-target inhibition of necroptotic signalling by established kinase inhibitors may contribute to their therapeutic efficacy in some contexts.

ABT-869 is a previously unreported inhibitor of necroptosis
To identify small molecules that modulate necroptosis at the level of MLKL or downstream of MLKL in the pathway, we performed a cell-based phenotypic screen using MDF cells expressing the MLKL Q343A mutant. In wild-type cells, necroptosis can be induced with the TSQ stimulus (TNF, T; Smac-mimetic Compound A, S; and pan-Caspase inhibitor Q-VD-OPh, Q) ( Figure 1A). In cells where an exogene encoding the MLKL Q343A mutant is inducibly expressed using doxycycline, MLKL Q343A triggers cell death in the absence of additional necroptotic stimulation ( Figure 1B) [36,37]. With this focused approach, a random selection of 5632 compounds from a diverse pharmacophore, in-house small molecule library and an additional 40 known kinase inhibitors available in-house were screened for their ability to inhibit cell death induced by the expression of this MLKL construct ( Figure 1C). The suppression of cell death by these inhibitors was monitored using CellTiter-Glo cell viability assays to enable high-throughput screening. From this screen, a small molecule within the kinase inhibitor subset, ABT-869 ( Figure 1D; Supplementary Figure S1A), was identified as an inhibitor of MLKL Q343A-induced death. ABT-869 was originally described as a multi-targeted receptor tyrosine kinase inhibitor developed by AbbVie that targets all members of the VEGF and PDGF receptor tyrosine kinase families [39]. ABT-869 attenuated necroptosis induced by expression of the MLKL Q343A constitutively active mutant in wild-type or Mlkl −/− MDF cells when applied at 1 mM concentration ( Figure 1E,F).
To validate ABT-869 as a hit from the screen, we performed cellular studies, which demonstrated that ABT-869 also inhibited necroptosis induced by TSQ treatment in wild-type MDF cells and displayed concentration-dependent inhibition, as monitored by propidium iodide (PI) uptake using flow cytometry ( Figure 1G). PI uptake provides a direct method of quantifying death at the level of each cell in a population, while CellTiter-Glo only offers a population-level assessment of viability. An analogue of ABT-869, WEHI-615 ( Figure 1D), lacking the terminal ring fluorine and methyl substituents, was subsequently synthesised to evaluate the specificity of ABT-869 as a necroptosis inhibitor. This analogue conferred less potent inhibition of TSQ-induced necroptosis than ABT-869 in wild-type MDF cells ( Figure 1G). We next performed a 5-point 3-fold titration from 10 mM of ABT-869 in wild-type MDF cells following stimulation with either TSQ (Figure 2A) or another necroptotic stimulus, TSZ, comprising TNF (T), Smac-mimetic Compound A (S) and the pan-Caspase inhibitor z-VAD-fmk (Z) ( Figure 2B). ABT-869 showed concentration-dependent inhibition of necroptotic cell death triggered by either TSQ or TSZ, as measured by PI uptake using flow cytometry. Interestingly, the protection was comparable between ABT-869 and two control necroptosis inhibitors, GSK 0 872 and GSK 0 843 (Supplementary Figure S1B) that target RIPK3 [40], at concentrations >1 mM when wild-type MDF cell death was induced with TSQ treatment (Figure 2A). In Part 1 of 2 (A) Schematic of the necroptosis pathway. TNF (T) activates TNFR1, the Smac-mimetic Compound A (S) blocks cIAP activity and the pan-caspase inhibitor Q-VD-OPh (Q) blocks caspase-8 activity. This TSQ stimulus results in activation of RIPK1 and RIPK3, and subsequent phosphorylation and contrast, when these cells were treated with TSZ, a more potent death stimulus than TSQ [41,42], we observed more potent protection with the control inhibitors compared with ABT-869 ( Figure 2B).

ABT-869 inhibits necroptosis in mouse and human cells
As ABT-869 was identified in a screen that used mouse cells expressing a constitutively active MLKL mutant and as our validation studies were performed in mouse cell lines, we further profiled the activity of ABT-869 and WEHI-615 in human cells. In U937 cells, a human lymphoma cell line commonly used to study necroptosis, we performed an 8-point 2-fold titration from 40 mM of ABT-869 and WEHI-615 where cell death was measured by PI uptake using IncuCyte live cell imaging following necroptosis induction. We used IncuCyte imaging to quantify cell death because this method allowed us to monitor the kinetics of cell death (PI uptake) over time. In contrast, using flow cytometry to quantify death by PI uptake only offers a snapshot of cell death at a fixed timepoint. ABT-869 blocked cell death when necroptosis was triggered with TSQ in U937 cells, and although variability between experiments made it difficult to accurately determine an IC 50 Figure S2F). TSI, comprising TS and the pan-Caspase inhibitor IDN-6556/Emricasan (I), is a more potent necroptosis trigger than TSQ or TSZ [35,41]. When U937 cells were stimulated with TSI, ABT-869 showed reduced inhibition of necroptosis, compared with TSQ stimulation, with an IC 50  ABT-869 binds to RIPK1, but not to RIPK3 or MLKL, in vitro and in cells Previously, we identified two necroptosis inhibitors using a similar screening strategy: the kinase inhibitor, AMG-47a, which directly targets RIPK1 and RIPK3 [37], and the HSP90 chaperone protein inhibitor, 17-AAG, which indirectly influences the folding of the HSP90 client proteins, RIPK1, RIPK3 and MLKL [36]. In light of these findings, and given that the core machinery required for TNF-dependent necroptosis comprises of two kinases (RIPK1, RIPK3) and a pseudokinase (MLKL) [27], we sought to establish if ABT-869, a kinase inhibitor, could also target these proteins to inhibit necroptosis. We first assessed the ability of ABT-869 and WEHI-615 to bind the necroptotic effectors in vitro using competition binding assays (DiscoverX KINOMEscan platform) ( Figure 3A), where compound binding to a given kinase or pseudokinase is determined through competition with an ATP-site directed probe [43]. ABT-869 and WEHI-615 bound to the human RIPK1 kinase domain with a K D of 1.6 mM and 8.0 mM, respectively. Neither compound bound the human RIPK3 kinase domain at any concentration tested (up to 30 mM). Interestingly, ABT-869 showed weak binding to full-length human MLKL with a K D of 12 mM, whereas WEHI-615 displayed stronger binding with a K D of 0.42 mM. As the IC 50 for ABT-869 inhibition of necroptotic cell death is estimated to be ∼5 mM in TSQ-treated U937 cells compared with the IC 50 of 35 mM for WEHI-615, and the ABT-869 binding affinity for RIPK1 is >7-fold higher than for MLKL, we hypothesised that targeting RIPK1, rather than MLKL, is likely to be the mechanism by which ABT-869 blocks cell death.
Next, we wanted to establish if ABT-869 and WEHI-615 interact with the necroptotic effector proteins in a cellular context. Using Cellular Thermal Shift Assays (CETSA) to evaluate cellular target engagement [44] Figure S1B) [45,46]. Both ABT-869 and WEHI-615 also increased the thermal stability of endogenous human RIPK1 in U937 cells, albeit not as profoundly as the established human RIPK1 inhibitor, GSK 0 481 (Supplementary Figure S1B) [47]. Neither ABT-869 nor WEHI-615 impacted the thermal stability of endogenous mouse RIPK3 in MDF cells or human RIPK3 in U937 cells. Interestingly, the established RIPK3 kinase inhibitor, GSK 0 872 [40], also had no effect on RIPK3 stability in MDF or U937 cells, consistent with previously published findings [37,48]. However, given that GSK 0 872 detectably binds recombinant RIPK3 kinase domain in vitro, while ABT-869 and WEHI-615 did not, it is likely that neither ABT-869 nor WEHI-615 bind to RIPK3 in cells. As in some cases compound binding fails to induce substantial change in the thermal stability of the target protein [44], we hypothesise this is the case for GSK 0 872 with RIPK3. Overall, the CETSA data suggest that ABT-869 interacts with RIPK1, but not RIPK3 or MLKL, in both mouse and human cells.
We then used Thermal Shift Assays (TSA) to further investigate the binding of ABT-869 and WEHI-615 to RIPK1 in vitro. In TSA, changes in the thermal stability of the target protein can be monitored using a fluorescent dye that binds hydrophobic residues, which become exposed as the protein unfolds upon increasing temperature [49].

ABT-869 inhibits RIPK1 kinase activity in vitro and in cells
To determine whether the binding of ABT-869 to RIPK1 impacts its kinase activity, we performed ADP-Glo Kinase Assays to evaluate RIPK1 autophosphorylation activity in vitro in the presence of ABT-869 or WEHI-615. Increasing concentrations of ABT-869 resulted in increasing inhibition of recombinant mouse RIPK1 kinase activity, with an IC 50 of 95 nM ( Figure 5A). WEHI-615 inhibited mouse RIPK1 kinase activity less potently, with an IC 50 of 1.6 mM ( Figure 5B). A similar trend was observed for human RIPK1, where ABT-869 inhibited recombinant human RIPK1 kinase activity with an IC 50 of 105 nM ( Figure 5C) and WEHI-615 with an IC 50 of 829 nM ( Figure 5D). In addition, neither compound inhibited recombinant mouse or human RIPK3 kinase activity, aside from some low level inhibition at the highest concentration tested (100 mM) ( Figure 5E-H). These results mirror the trends observed in the TSA binding data, and demonstrate that ABT-869 binds to and inhibits RIPK1, but not RIPK3, in vitro.
We then assessed the influence of ABT-869 on RIPK1 kinase activity in cells. TNF-driven necroptosis induction results in the autophosphorylation of RIPK1 and RIPK3, which are key events in this cell death signalling  pathway [22,[50][51][52][53][54][55]. Initially, we performed time course experiments to map the chronology of mouse RIPK1 and RIPK3 autophosphorylation events in MDF cells over a 4 h period following necroptosis induction (Supplementary Figure S5A,B). By Western blot, we detected autophosphorylation of S166 in the RIPK1 activation loop at 1 h, which was maintained at 2 h and decreased at 3 and 4 h. Autophosphorylation of the RIPK3 C-lobe residues, T231/S232, was observed at 2 h, with levels maintained at 3 h and 4 h. Total RIPK1 and RIPK3 levels remained constant over this 4 h time period following TSI stimulation.
As maximal RIPK1 autophosphorylation was observed 2 h post-TSI stimulation, MDF cells treated with ABT-869, WEHI-615 and control inhibitors were stimulated with TSI for 2 h and phospho-protein levels were determined by Western blot ( Figure 5I; Supplementary Figure S5C). ABT-869 at 10 mM almost completely ablated RIPK1 phosphorylation, to a similar extent as the control RIPK1 inhibitor, Nec-1s, whereas WEHI-615 only partially reduced the phospho-RIPK1 signal at 10 mM. Similarly, the phospho-RIPK3 signal observed following 2 h TSI stimulation was almost completely inhibited by 10 mM ABT-869, to a similar extent to the control RIPK3 kinase inhibitor, GSK 0 872, while 10 mM WEHI-615 only partially reduced RIPK3 phosphorylation. However, given that ABT-869 did not bind to RIPK3 in vitro or in cells and did not inhibit RIPK3 kinase activity in vitro, the observed inhibition of RIPK3 autophosphorylation in cells is most likely attributable to the upstream inhibition of RIPK1 by ABT-869, which would prevent the activation and autophosphorylation of RIPK3. Together, these data demonstrate that ABT-869 inhibits RIPK1 kinase activity in vitro and in cells.

Discussion
In a cell-based phenotypic screen for small molecules that block the killing mediated by an activated form of the necroptotic executioner, MLKL, we identified the kinase inhibitor, ABT-869 (Linifanib). Although ABT-869 was developed as a receptor tyrosine kinase inhibitor, here we identified the RIPK1 kinase as the target underpinning the ability of ABT-869 to inhibit necroptosis. ABT-869 blocked necroptotic signalling in mouse and human cell lines treated with a necroptotic stimulus cocktail (TSQ, TSZ or TSI), and death induced upon expression of an activated mutant form of mouse MLKL. RIPK1 was validated as the target of ABT-869 in cells using CETSA and immunoblots for necroptosis effector phosphorylation, in DiscoverX KINOMEscan competition assays, and thermal shift and enzymatic assays using recombinant proteins. Consistent with our findings, Yu et al. [56] in a very recent parallel study, validated ABT-869 as an inhibitor of the necroptosis pathway, which could target RIPK1 to confer protection in mice from TNF-induced sterile sepsis.
RIPK1 is typically considered to be the apical kinase in the necroptosis pathway, which acts upstream of RIPK3 and MLKL in directing cell death, following exposure to a death receptor ligand or pathogen molecular pattern. Recent data, however, have implicated RIPK1 as serving an important function downstream of MLKL activation [37], likely in scaffolding the assembly of the high molecular weight cytoplasmic platform termed the necrosome. Our findings provide further support for this idea, because cell death mediated by a constitutively activated form of MLKL could be inhibited by ABT-869, which targets RIPK1, but not RIPK3 or MLKL, implicating RIPK1 as performing a function in signalling subsequent to MLKL activation ( Figure 6).
Our data add to a growing repertoire of tyrosine kinase inhibitors that target RIPK1 to block necroptosis via off-target mechanisms. Recently, an FGF receptor inhibitor, AZD4547, was reported to potently inhibit necroptosis by targeting RIPK1 [57]. This adds to several other tyrosine kinase inhibitors (reviewed in [58]), which includes sunitinib [59], pazopanib [59], ponatinib [60], and AMG-47a [37], now known to bind and inhibit RIPK1 in an off-target manner. Broadly speaking, this raises the prospect that other clinically approved kinase inhibitors may also act synergistically on the necroptosis pathway. The extent to which this binding may ameliorate inflammatory signalling by dampening necroptotic cell death and thus impact the clinical efficacy remains of outstanding interest.  Our findings underscore the challenges associated with identifying compounds that interfere with the terminal steps in necroptosis signalling using cell-based screens. RIPK1 appears to play two critical roles in the necroptosis pathway: (i) upon autophosphorylation, RIPK1 scaffolds the assembly of the necrosome with RIPK3; and (ii) RIPK1-scaffolded necrosome assembly is required to connect activated MLKL to the trafficking machinery to enable plasma membrane translocation and lytic cell death. These upstream and downstream signalling roles of RIPK1 in the necroptosis pathway rationalise the prevalence of RIPK1 inhibitors identified in cell-based screens as pathway inhibitors. Similar approaches have successfully identified inhibitors that target RIPK1 and RIPK3 kinase activity [37], and the HSP90 chaperone [36], which is critical to RIPK1, RIPK3 and MLKL stability, in cell-based screens. Future efforts will be important to identify inhibitors that target the terminal steps in the pathway at the level of MLKL oligomerisation and/or membrane association, or downstream at the level of as-yet-unknown (co)effectors.

Phenotypic screen
The phenotypic screen was performed as previously described [36,37].  Assays. Increasing concentrations of ABT-869 or WEHI-615 were tested for their ability to inhibit the autophosphorylation (IC 50 ) cell viability was determined using the CellTiter-Glo Assay (Promega) by measuring luminescence using an EnVision plate reader (PerkinElmer). For the 40 kinase inhibitors, WT and Mlkl −/− MDF cells expressing MLKL Q343A were seeded at 5.0 × 10 3 cells per well in 96-well plates and allowed to settle for ∼6 h at 37°C 10% CO 2 . Cells were treated with the kinase inhibitors (1 mM) for 1 h and then expression of the MLKL Q343A protein was induced with doxycycline (1 mg/ml). After 18 h, cell viability was determined using the CellTiter-Glo Assay (Promega) by measuring luminescence using an EnVision plate reader (PerkinElmer). Raw luminescence data were normalised to the uninduced (no doxycycline) controls (100% cell viability) using GraphPad Prism.

FACS cell death assays
FACS cell death assays were performed as previously described [37,48] using two protocols. In the first protocol, WT MDF cells were seeded at 1.0 × 10 5 cells per well in 24-well plates and allowed to settle for 4 h at 37°C 10% CO 2 . Cells were treated with ABT-869 (0.2 mM, 0.6 mM, 1 mM, 5 mM), WEHI-615 (0.2 mM, 0.6 mM, 1 mM, 5 mM) or DMSO alone and Q-VD-OPh (10 mM) for 30 min before addition of TNF (100 ng/ml) and Smac mimetic (Compound A; 500 nM). After 24 h, cells were harvested, stained with 1 mg/ml propidium iodide (PI; Sigma-Aldrich) and PI positive cells were quantified by flow cytometry on a BD FACSCalibur instrument. In the second protocol, WT MDF cells were seeded at 5.0 × 10 4 cells in 500 ml per well in 24-well plates (Falcon, cat #353047) and allowed to settle for 24 h at 37°C 10% CO 2 . Cells were treated with ABT-869 (5-point 3-fold titration from 10 mM), GSK 0 872 (1 mM or 10 mM), GSK 0 843 (1 mM or 10 mM), DMSO alone or left untreated for 1 h. Cells were then stimulated with TSQ or TSZ for 24 h to induce necroptosis, or left unstimulated. For all compounds and reagents, 0.5 ml of 1000× stocks were used. Cells were harvested, stained with 2 mg/ml propidium iodide (PI; Sigma-Aldrich) and PI positive cells were quantified by flow cytometry on a BD FACSCalibur instrument.

Discoverx KINOMEscan binding assays
Binding affinities (K D ) of ABT-869 and WEHI-615 for human MLKL, RIPK1 and RIPK3 were obtained from DiscoverX KINOMEscan, a commercial kinase inhibitor binding platform, using the KdELECT service [43]. Briefly, active/pseudoactive site competition binding assays were performed by measuring compound binding to a given kinase/pseudokinase through competition with a promiscuous probe. The ability of ABT-869 and WEHI-615 to compete with this immobilised, active-site directed ligand was quantitatively measured via qPCR of the DNA tagged-protein. ABT-869 and WEHI-615 were tested in an 11-point 3-fold dilution series against human MLKL (full-length), RIPK1 (kinase domain) and RIPK3 (kinase domain) to obtain K D values.

Cellular thermal shift assays
CETSA was performed as previously described [37,48] Samples were freeze-thawed once to lyse the cells. Soluble protein was separated from precipitated protein via centrifugation at 17 000×g for 30 min at 4°C. Soluble protein fractions were resolved by SDS-PAGE and analysed by Western blot. Briefly, 10 ml of each soluble protein sample was added to 10 ml of NuPAGE LDS Sample Buffer (Invitrogen) containing TCEP (Sigma-Aldrich). Samples were heated at 95°C for 10 min and then 15 ml of each sample was run on a gel along with the Precision Plus Kaleidoscope Prestained Protein Standard (Bio-Rad), using either NuPAGE 4-12% Bis-Tris 1.0 mm × 20-well Midi Gels (Invitrogen) with NuPAGE MES SDS Running Buffer (Invitrogen), or 4-15% Criterion TGX Stain-Free 26 well 15 ml Protein Gels (Bio-Rad) with Tris/Glycine Buffer (Bio-Rad). Western blots were performed according to the Western Blotting General Procedure above.

Data Availability
All data and reagents are available from the authors upon request. Uncropped Western blots are included as supplementary data.

Ethics Approval
Mouse dermal fibroblasts were prepared using procedures approved by and conducted in accordance with the Animal Ethics Committee of the Walter and Eliza Hall Institute, Australia.